Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07G—COMPOUNDS OF UNKNOWN CONSTITUTION
  • C07G1/00—Low-molecular-weight derivatives of lignin
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/02—Monosaccharides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis

Definitions

• the present invention relates to a method for producing a polyphenol-containing composition from herbaceous biomass.
• Cellulose-containing biomass is mainly composed of cellulose and hemicellulose, which are polysaccharides, and lignin, which is an aromatic polymer. A decomposed liquid can be obtained.
• Patent Document 1 describes a method for efficiently obtaining hydroxycinnamic acid by passing an alkaline aqueous medium through cellulose-containing biomass
• Patent Document 2 describes an acid-insoluble A method for recovering lignin is described.
• Patent Document 3 an extract obtained by treating bagasse, which is sugar cane strained lees, with an alkaline solution is adjusted to acidity and filtered, and the filtrate is adsorbed with an aromatic synthetic adsorbent to efficiently remove polyphenols.
• a method of making the composition is described.
• the decomposition solution containing polyphenols obtained by the above-described conventional technology can be used as a deodorant (Patent Document 4), a discoloration inhibitor for food (Patent Document 5), an aquatic organism growth promoter (Patent Document 6), and the like.
• a deodorant Patent Document 4
• a discoloration inhibitor for food Patent Document 5
• an aquatic organism growth promoter Patent Document 6
• coumaric acid and ferulic acid are contained as representative polyphenols.
• one object of the present invention is to provide a new technical means for efficiently producing a polyphenol-containing composition from herbaceous biomass.
• a polyphenol-containing composition can be efficiently produced from herbaceous biomass by adjusting the pH of the extract obtained by treating herbaceous biomass with alkali and reacting it with a specific enzyme. , have completed the present invention.
• [1] A method for producing a polyphenol-containing composition derived from herbaceous biomass, (1) a step of contacting the herbaceous biomass with an alkaline aqueous solution to obtain an extract; (2) a step of adjusting the extract obtained in step (1) to pH 3.2 or higher and 4.5 or lower and reacting it with an enzyme containing cellobiohydrolase activity and xylanase activity to obtain an enzyme reaction solution; ) a step of solid-liquid separation of the enzymatic reaction solution obtained in step (2) to obtain a clarified solution containing a polyphenol-containing composition;
• a method for producing a polyphenol-containing composition comprising: [2] The method for producing a polyphenol-containing composition according to [1], wherein the extract obtained in step (1) is adjusted to pH 3.5 or more and less than 4.0.
• a polyphenol-containing composition can be efficiently produced from herbaceous biomass. Moreover, according to the present invention, when a polyphenol-containing composition is produced from herbaceous biomass, it is possible to prevent micellization-like phenomena and improve the efficiency of solid-liquid separation. Moreover, according to the present invention, the filterability of the product can be improved even in the separation membrane treatment after the solid-liquid separation.
• a method for producing a herbaceous biomass-derived polyphenol-containing composition comprises: (1) a step of contacting the herbaceous biomass with an alkaline aqueous solution to obtain an extract; (2) a step of adjusting the extract obtained in step (1) to pH 3.2 or higher and 4.5 or lower and reacting it with an enzyme containing cellobiohydrolase activity and xylanase activity to obtain an enzyme reaction solution; ) a step of solid-liquid separation of the enzymatic reaction solution obtained in step (2) to obtain a clarified solution containing a polyphenol-containing composition; is characterized by including
• step (1) herbaceous biomass is brought into contact with an alkaline aqueous solution to obtain an extract.
• examples of herbaceous biomass include sugarcane pomace bagasse, switchgrass, napiergrass, erianthus, corn stover, corn hulls, wheat husks, soybean hulls, rice straw, wheat straw. , oil palm empty fruit bunches, etc., but not limited to these.
• herbaceous biomass with a lignin content of 5% or more.
• bagasse, napier grass, erianthus, corn stover, and rice straw are preferable, and bagasse is more preferable.
• the lignin content can be determined by measuring Clason lignin, which is obtained by subtracting ash from the acid hydrolysis residue.
• the shape of the herbaceous biomass is not particularly limited, but it is preferable that the herbaceous biomass is pulverized.
• the pulverization means is not particularly limited, and machines commonly used for coarse pulverization of various materials such as ball mills, vibration mills, cutter mills, hammer mills, Willey mills and jet mills can be used. This mechanical pulverization may be either dry or wet, preferably dry pulverization.
• the water content of the herbaceous biomass is not particularly limited, but a preferable range is, for example, about 3% or more, about 3% or more and about 75% or less, about 5% or more, and about 5%.
• a preferable range is, for example, about 3% or more, about 3% or more and about 75% or less, about 5% or more, and about 5%.
• polyphenols may include one or more of hydroxycinnamic acids such as coumaric acid and ferulic acid, and lignin degradation products, and can be measured, for example, by the Folin-Ciocalteu method.
• the Folin-Ciocalteu method was originally developed for the purpose of analyzing aromatic amino acids such as tyrosine and tryptophan, and proteins containing them.
• the phenolic hydroxyl group is alkaline and the blue color produced by reduction of phosphotungstic acid and molybdic acid is colorimetrically determined at 700 to 770 nm.
• a similar operation is performed using a specific reference substance such as gallic acid or catechin, and a quantitative value can be shown in terms of the compound, and the value in terms of catechin is used in the present invention.
• the alkaline aqueous solution is ammonia, aqueous ammonia, alkali metal hydroxides, alkali metal oxides, alkaline earth metal oxides, alkali metal carbonates, alkaline earth metal carbonates, water
• An alkaline aqueous solution containing at least one selected from quaternary ammonium oxide and the like can be mentioned, preferably an aqueous medium containing at least one hydroxide selected from sodium hydroxide and potassium hydroxide, more preferably sodium hydroxide solution or potassium hydroxide solution.
• the alkali concentration of the alkaline aqueous solution is not particularly limited, but is 0.05% by weight or more and 10% by weight or less, preferably 0.05% by weight or more and 5% by weight or less, more preferably 0.1% by weight or more and 5% by weight or less. is 0.1% by weight or more and 3% by weight or less, and 0.1% by weight or more and 2% by weight or less.
• the lower limit of the pH of the alkaline aqueous solution is not particularly limited as long as it is alkaline, but is pH 7 or higher, preferably pH 8 or higher, more preferably pH 9 or higher, and still more preferably pH 10 or higher.
• the upper limit of the pH is not particularly limited as long as it is less than pH 14, but from the viewpoint of reducing the amount of alkali used, it can be set at pH 12 or less.
• a preferable pH range is, for example, 7 or more and 13.5 or less, 8 or more and 13.5 or less, a more preferable pH range is 9 or more and 13.5 or less, and a further preferable pH range is 10 or more and 12 or less. be.
• the temperature at which the alkaline aqueous solution and herbaceous biomass are brought into contact is 60°C or higher.
• a pressure exceeding normal pressure to the alkali-treated product, which requires high-pressure equipment. More preferably 80 to 100°C, more preferably 60°C to less than 100°C, and 80°C to less than 100°C.
• the weight ratio of the alkaline aqueous solution and the herbaceous biomass is not particularly limited, but the preferred range is, for example, 100:1 to 2:1, 90: 1-3:1, 50:1-5:1, 30:1-5:1, 25:1-7:1, 25:1-7:1, 25:1-5:1, 20:1- 5:1.
• the method of contacting the herbaceous biomass with the alkaline aqueous solution is not particularly limited, but examples thereof include a method of contacting the herbaceous biomass with the alkaline aqueous solution by spraying, immersing, or passing a liquid through the herbaceous biomass. It may be stirred or the container may be rotated so that the herbaceous biomass and the herbaceous biomass are in sufficient contact with each other.
• the contact time between the alkaline aqueous solution and the herbaceous biomass is not particularly limited, but is preferably about 20 minutes or more and about 72 hours or less, about 20 minutes or more and about 48 hours or less, about 20 minutes or more and about 24 hours or less, or about 30 minutes or more. About 48 hours or less, about 30 minutes or more and about 24 hours or less, about 30 minutes or more and about 12 hours or less, about 30 minutes or more and about 6 hours or less, or about 30 minutes or more and about 3 hours or less.
• An extract can be obtained by solid-liquid separation of the herbaceous biomass and the alkaline aqueous solution.
• solid-liquid separators include screw presses and centrifuges. A strainer or the like may be used to remove fine particles.
• the liquid after passing may be used as it is as an extract liquid, but the reactant of the herbaceous biomass is separated by a solid-liquid separation device. Squeezing is preferable from the viewpoint of recovery of the extract
• step (2) the extract obtained in step (1) is adjusted to pH 3.2 or more and 4.5 or less, and an enzyme containing cellobiohydrolase activity and xylanase activity to obtain an enzyme reaction solution.
• an acidic substance is added to the extract obtained in the step of obtaining the extract to adjust the pH to the acidic range as described above.
• Acidic substances include, but are not limited to, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, lactic acid, acetic acid, formic acid, citric acid, etc., but are preferably hydrochloric acid, sulfuric acid, nitric acid, and more preferably hydrochloric acid.
• the method of adjusting the pH using an acidic substance is not particularly limited, but an example is a method of adding and mixing an appropriate concentration of an acidic substance while checking the pH.
• a continuous system in which the alkaline extract is continuously added during pH adjustment and the liquid after pH adjustment is continuously withdrawn, or a batch system may be used.
• the temperature during pH adjustment is not particularly limited, it is 20°C or higher and 100°C or lower, preferably 20°C or higher and 60°C or lower, more preferably 30°C or higher and 60°C or lower.
• the pH adjustment range is usually 3.2 or more and 4.5 or less, preferably pH 3.3 or more and 4.5 or less, more preferably pH 3.3 or more and less than 4.0, more preferably pH 3.5 or more.
• the pH is 4.0 or less, more preferably in the range of pH 3.5 or more and less than 4.0.
• An enzyme with cellobiohydrolase activity is an exo-enzyme that produces cellobiose by degrading the cellulose chain with ⁇ -1,4-linked glucose from the end.
• the cellobiohydrolase activity can be measured as an enzymatic activity that decomposes 4-nitrophenyl- ⁇ -D-lactopyranoside.
• the amount of enzyme that produces 1 ⁇ mol of 4-nitrophenol per minute is defined as 1 U.
• Enzyme activity is measured by a method according to the procedure described in Reference Example 2 below.
• an enzyme having a cellobiohydrolase activity of 5 U/g or more is defined as an enzyme containing cellobiohydrolase activity. 1000 U/g, more preferably 5 to 500 U/g, still more preferably 10 to 500 U/g, particularly preferably 20 to 300 U/g.
• Enzymes with xylanase activity are endo-type enzymes that randomly degrade xylans with ⁇ -1,4-linked xylose.
• Xylanase activity can be determined by measuring the amount of reducing sugar contained in the reaction solution after the reaction using a commercially available xylan reagent (e.g., Birchwood xylan) as a substrate. Kit (XylX6 method)” is preferably used.
• the XylX6 reagent is decomposed by a combination of the xylanase in the measurement target and the auxiliary reagent ⁇ -xylosidase to generate 4-nitrophenol, thereby measuring xylanase activity. be able to.
• the amount of enzyme that produces 1 ⁇ mol of 4-nitrophenol per minute is defined as 1 U.
• the enzymatic activity is measured according to the procedure described in Reference Example 3 below.
• an enzyme having a xylanase activity of 400 U/g or more is defined as an enzyme containing xylanase activity.
• the xylanase activity value is preferably 400 to 50000 U/g, more preferably is 500 to 50,000 U/g, more preferably 1,000 to 50,000 U/g, particularly preferably 3,000 to 45,000 U/g.
• the enzyme is produced by a microorganism, for example, it may be produced by a single microorganism or by a plurality of microorganisms.
• Microorganisms that produce cellobiohydrolase and xylanase include Trichoderma, Aspergillus, Cellulomonas, Clostridium, Streptomyces, and Humicola. , Acremonium, Irpex, Mucor, Talaromyces, and the like, preferably Trichoderma.
• Trichoderma microorganism is not particularly limited, Trichoderma reesei is preferred, and specifically Trichoderma reesei QM9414, Trichoderma reesei QM9123, Trichoderma reesei RutC-30 reeseiRut C-30), Trichoderma reesei PC3-7, Trichoderma reesei CL-847, Trichoderma reesei MCG77, Trichoderma reesei MCG80, Trichoderma reesei MCG80 (Trichoderma reesei MCG80) Trichoderma viride QM9123 (Trichoderma viride9123) can be exemplified.
• the microorganisms derived from the genus Trichoderma described above may be mutant strains that have been subjected to mutation treatment with a mutating agent or ultraviolet irradiation to improve cellulase productivity.
• the enzyme may be added in a purified form, or may be added as a crude enzyme in the culture medium, as long as the enzyme satisfies the activity described above.
• Agents may be used and may include enzymes other than cellobiohydrolases and xylanases.
• ⁇ -glucosidase, ⁇ -xylosidase, endoglucanase, mannanase and the like may be included.
• cellulase agents and xylanase agents include, for example, Novozyme's "Selic-Seetech” (registered trademark) and “Seric-Seetech 2" (registered trademark), and Danisco Japan's “Accellace” (registered trademark) 1000. , “Accel Race” (registered trademark) 1500, “Accel Race” (registered trademark) Duet, and Sigma-Aldrich's "Cellulase from Trichoderma reesei ATCC 26921", “Cellulase from Trichoderma viride", and “Cellulase from Trichoderma longibrachiatum”. and “Cellulosine TP25" and “Cellulosine HC100” of HI Co., Ltd., but are not limited thereto.
• the amount of the enzyme to be added may be changed as appropriate depending on the enzyme to be added, and is not particularly limited. parts by weight, preferably 0.005 to 20 parts by weight, more preferably 0.005 to 5 parts by weight.
• Adjusting the extract to a pH within the predetermined range and reacting it with the enzyme to obtain an enzyme reaction liquid means an enzymatic reaction in which the enzyme is present in the liquid with the extract adjusted to a pH within the predetermined range.
• the enzyme may be added during pH adjustment, but it is preferable to add the enzyme after adjusting the pH to within a predetermined range. Addition of the above enzyme may be carried out in a continuous manner or in a batch manner.
• the reaction time for the enzyme is the time for the enzyme to exist in a pH state within the predetermined range and for solid-liquid separation to obtain a clear solution. In the case of a continuous system, it is the retention time during which the enzyme is present at the pH within the predetermined range and the solid-liquid separation process is performed to obtain a clarified liquid.
• the time for which the enzyme is allowed to react at the pH within the predetermined range is not particularly limited, but is preferably 5 minutes to 8 hours, more preferably 5 minutes to 6 hours, even more preferably 5 minutes to 4 hours, and still more. It is preferably 10 minutes or more and 4 hours or less, particularly preferably 10 minutes or more and 2 hours or less.
• the temperature at which the enzyme is reacted may be appropriately changed according to the enzyme used, and is not particularly limited, but is preferably 15 to 100°C, more preferably 30 to 60°C, and even more preferably 35 to 55°C.
• step (3) the enzymatic reaction solution obtained in step (2) is subjected to solid-liquid separation to obtain a clarified liquid containing a polyphenol-containing composition.
• the method of solid-liquid separation treatment is not particularly limited, and methods such as filtration, centrifugation, and sedimentation separation can be used, and combinations thereof can also be used.
• a sedimentation separator a decanter-type centrifuge, a plate-type centrifuge, a screw press, a filter press, a belt press, a belt screen, or the like can be used, and a combination thereof can also be used.
• a filter press, a decanter type centrifuge, and a disc plate type centrifuge are preferred.
• a filter aid may be used depending on the solid-liquid separation method.
• Filter aids include diatomaceous earth, perlite, cellulose, activated carbon and the like, but are not limited to these, but diatomaceous earth is preferred.
• the filter aid may be added from the step of obtaining the extract until the solid-liquid separation treatment of the enzymatic reaction solution is performed to obtain the clarified solution, and the timing of addition is not particularly limited.
• the amount of the filter aid is not particularly limited, but is 0.05 to 10 parts by weight, preferably 0.1 to 5 parts by weight, per 100 parts by weight of the enzyme reaction solution.
• the clarified liquid obtained in the above step (3) is selected from the group consisting of microfiltration membranes, ultrafiltration membranes, nanofiltration membranes and reverse osmosis membranes. Filtration through the above separation membrane yields a permeate and/or non-permeate containing the polyphenol-containing composition.
• the permeated liquid obtained by filtering the clarified liquid through the microfiltration membrane in step (4) is filtered from the ultrafiltration membrane, the nanofiltration membrane and the reverse osmosis membrane. Filtration through one or more separation membranes selected from the group consisting of recovering the retentate containing the polyphenol-containing composition.
• the clarified liquid or the permeated liquid obtained by filtering through a microfiltration membrane in step (4) is filtered through a nanofiltration membrane to obtain a polyphenol-containing composition.
• a permeate solution containing substances is obtained.
• a microfiltration membrane is a membrane with an average pore size of 0.01 ⁇ m to 5 mm, and is abbreviated as microfiltration or MF membrane.
• the average pore size is preferably 0.45 ⁇ m or less, more preferably 0.22 ⁇ m or less.
• the material of the microfiltration membrane is not particularly limited, but examples include celluloses, aromatic polyamides, polyvinyl alcohol, polysulfone, polyvinylidene fluoride, polyethylene, polyacrylonitrile, polypropylene, polycarbonate, polytetrafluoroethylene, ceramics, metals, and the like. can be used.
• the form of the microfiltration membrane includes, but is not limited to, a hollow fiber type, a tubular type, a flat membrane type, a spiral type, and the like.
• the filtration method may be dead end filtration, cross flow filtration, constant pressure filtration, or constant flow filtration. If the microfiltration membrane is clogged, it can be reverse washed by passing the washing liquid from the permeate side of the membrane to the non-permeate side, or by supplying gas to the non-permeate side of the membrane to remove the cake formed on the membrane surface. You may perform air washing to let you do it. Washing liquids for backwashing include filtrates from microfiltration membranes, water, and chemical solutions.
• An ultrafiltration membrane is a membrane with a molecular weight cutoff of more than 1,000 and 200,000 or less, and is abbreviated as ultrafiltration, UF membrane, etc.
• the ultrafiltration membrane has a pore size that is too small to measure the pore size on the membrane surface with an electron microscope or the like. It is supposed to be Membrane Science Experiment Series Vol. , the data plotted with the rejection on the vertical axis is called a molecular weight cutoff curve. The molecular weight at which the rejection rate is 90% is called the cut-off molecular weight of the membrane. ], it is well known to those skilled in the art as an index representing the membrane performance of an ultrafiltration membrane.
• the material of the ultrafiltration membrane is not particularly limited, but examples include celluloses, aromatic polyamides, polyvinyl alcohol, polysulfone, polyvinylidene fluoride, polyethylene, polyacrylonitrile, polypropylene, polycarbonate, polytetrafluoroethylene, ceramics, and metals. etc. can be used.
• the material of the ultrafiltration membrane is not particularly limited, but examples include celluloses, aromatic polyamides, polyvinyl alcohol, polysulfone, polyvinylidene fluoride, polyethylene, polyacrylonitrile, polypropylene, polycarbonate, polytetrafluoroethylene, ceramics, and metals. etc. can be used.
• the form of the ultrafiltration membrane includes, but is not limited to, a hollow fiber type, a tubular type, a flat membrane type, a spiral type, and the like.
• the filtration method may be dead end filtration, cross flow filtration, constant pressure filtration, or constant flow filtration.
• the nanofiltration membrane is also called a nanofilter (nanofiltration membrane, NF membrane), and is a membrane generally defined as "a membrane that allows monovalent ions to pass through and blocks divalent ions”. . It is a membrane that is thought to have minute voids of several nanometers, and is mainly used to block minute particles, molecules, ions, salts, etc. in water.
• Polymer materials such as cellulose acetate-based polymers, polyamides, polyesters, polyimides, and vinyl polymers can be used as materials for the nanofiltration membrane, and membranes containing multiple membrane materials can also be used.
• the form of the nanofiltration membrane includes, but is not limited to, a hollow fiber type, a tubular type, a flat membrane type, a spiral type, and the like.
• the filtration method may be dead end filtration, cross flow filtration, constant pressure filtration, or constant flow filtration.
• the molecular weight cut off of the nanofiltration membrane is usually 100 to 1,000, preferably 150 to 1,000, more preferably 300 to 1,000.
• a nanofiltration membrane having a molecular weight cutoff of 300 or more and 1000 or less By filtering with a nanofiltration membrane having a molecular weight cutoff of 300 or more and 1000 or less, the colored substances in the polyphenol component are concentrated on the non-permeate side, and the polyphenol component with reduced coloring can be obtained on the permeate side.
• a polyphenol-containing composition as permeate or non-permeate liquid of the separation membranes. can be recovered, it is preferable to recover the polyphenol-containing composition as a permeate of the separation membrane.
• clogging of the ultrafiltration membrane or the nanofiltration membrane can be prevented by filtering the clarified liquid with the microfiltration membrane before filtering through the ultrafiltration membrane or the nanofiltration membrane.
• the components contained in the clarified liquid may be fractionated and/or purified by combining ultrafiltration membranes and/or nanofiltration membranes with different pore sizes.
• a reverse osmosis membrane is also called an RO membrane, and is generally defined as "a membrane that has a desalting function including monovalent ions”. This membrane is thought to have ultra-fine pores of several angstroms to several nanometers in size, and is mainly used to remove ion components in seawater desalination and ultrapure water production. In addition, the cutoff molecular weight is less than 100.
• the material of the reverse osmosis membrane can be a polymer material such as cellulose acetate-based polymer, polyamide, polyester, polyimide, vinyl polymer, etc., and the membrane includes a plurality of membrane materials.
• a polymer material such as cellulose acetate-based polymer, polyamide, polyester, polyimide, vinyl polymer, etc.
• the membrane includes a plurality of membrane materials.
• Examples of the form of the reverse osmosis membrane include, but are not limited to, hollow fiber type, tubular type, flat membrane type, and spiral type.
• the filtration method may be dead end filtration, cross flow filtration, constant pressure filtration, or constant flow filtration.
• the above clarified liquid can be filtered through a reverse osmosis membrane to concentrate the polyphenol components on the non-permeate side.
• a reverse osmosis membrane depending on the type of reverse osmosis membrane and operating conditions, it is possible to remove impurities other than polyphenol components to the permeation side and refine while concentrating the polyphenol components. That is, when the clarified liquid is filtered through a reverse osmosis membrane, the polyphenol-containing composition is recovered as a non-permeate liquid of the separation membrane.
• the clarified liquid is filtered through one or more separation membranes selected from the group consisting of microfiltration membranes, ultrafiltration membranes and nanofiltration membranes, and the permeated liquid is filtered through a reverse osmosis membrane to A polyphenol-containing composition may be recovered as a permeate.
• the non-reverse osmosis membrane By filtering the permeate obtained by filtering through the microfiltration membrane and / or ultrafiltration membrane through the reverse osmosis membrane, while preventing clogging of the reverse osmosis membrane, the non-reverse osmosis membrane
• the polyphenol component can be concentrated on the permeate side, it is preferable to filter at least the permeate filtered through the microfiltration membrane with a reverse osmosis membrane to concentrate the polyphenol component on the non-permeate side of the reverse osmosis membrane.
• the polyphenol component may be fractionated or purified by filtering the permeated liquid of the nanofiltration membrane through a reverse osmosis membrane.
• a herbaceous biomass-derived polyphenol-containing composition can be obtained by the production method described above.
• the herbaceous biomass-derived polyphenol-containing composition is characterized by containing at least coumaric acid as a polyphenol component and xylose as a component other than polyphenols.
• the composition ratio of these components is not particularly limited, but the content of xylose relative to coumaric acid (% (w/w)) is preferably 1 to 200% (w/w), more preferably 2 to 100% (w/w). w), more preferably 2 to 50% (w/w), an effect that coloration does not easily progress over time can be obtained.
• the degree of coloring can be evaluated by absorbance at 550 nm, and the lower the absorbance, the lower the degree of coloring.
• the polyphenol-containing composition may be in the form of liquid or powder. It is preferable in terms of obtaining the effect of not being sticky when touched by hand. Although the method for making powder is not particularly limited, spray drying is preferred.
• JIS Japanese Industrial Standards
• a small filter device MO-4 manufactured by Yabuta Sangyo Co., Ltd. was used as a filter press.
• solid-liquid separation by a filter press was carried out in the same manner, except that no enzyme was added to the alkali-treated liquid adjusted to pH 3.5.
• Table 3 shows the results of the treatment for 5 minutes.
• Example 7 As shown in Table 3, in Examples 7 and 8, compared to Comparative Example 7, the amount of filtrate after 5 minutes was larger, and the solid-liquid separation performance by the filter press was improved. In addition, the amount of filtrate was larger in Example 7 than in Example 8, and the solid-liquid separation property in the filter press was improved.
• Example 9 in which the enzyme was added to the extract adjusted to pH 3.5, the filterability with the microfiltration membrane was significantly improved compared to the case where the enzyme was not added. On the other hand, when the enzyme was added at pH 5.0, there was almost no change in filterability with the microfiltration membrane.
• the permeated liquids of the microfiltration membranes are ultrafiltration membrane: "SPE50” (manufactured by Synder, molecular weight cutoff 50,000 Da), nanofiltration membrane 1: “SPE1” (manufactured by Synder, molecular weight cutoff 1,000 Da ), nanofiltration membrane 2: “NFG” (manufactured by Synder, molecular weight cutoff 600 to 800 Da), nanofiltration membrane 3: “NFW” (manufactured by Synder, molecular weight cutoff 300 to 500 Da), reverse osmosis membrane: “BW60 -1812-75” (obtained from Filmtec module, salt rejection rate 99%).
• the microfiltration membrane was filtered in the same manner even under the condition of no enzyme addition, and the permeated liquid of the microfiltration membrane was filtered with the same type of membrane.
• the permeated liquids of the microfiltration membranes are, respectively, nanofiltration membrane 2: “NFG” (manufactured by Synder, molecular weight cutoff 600 to 800 Da), nanofiltration membrane 3: “NFW” (manufactured by Synder, molecular weight cutoff 300 to 500 Da ), reverse osmosis membrane: "BW60-1812-75” (obtained from Filmtec module, salt rejection rate 99%).
• NFG nanofiltration membrane 2
• nanofiltration membrane 3 “NFW” (manufactured by Synder, molecular weight cutoff 300 to 500 Da )
• reverse osmosis membrane "BW60-1812-75” (obtained from Filmtec module, salt rejection rate 99%).
• constant flow rate filtration is performed at a membrane surface linear velocity of 20 cm / sec and a filtration flux of 0.2 m / D, and the operating pressure is 6 MPa as the upper limit, and when 6 MPa is reached, the constant pressure is 6 MPa. Switch to filtration and concentrate
• the permeated liquid of the microfiltration membrane, the permeated liquid of the nanofiltration membranes 2 and 3, and the non-permeated liquid of the reverse osmosis membrane were visually evaluated for the presence or absence of coloring, and in order to quantify it, measurement was performed with a spectrophotometer. . Measurements were made immediately after preparation and 7 days after preparation and stored in a refrigerator. The absorbance of the solution was measured using a spectrophotometer UV-1280 (manufactured by Shimadzu Corporation) with a wavelength of 550 nm, which was a peak characteristic of this chromaticity measurement difference, as an indicator.
• the permeated liquid of the microfiltration membrane, the permeated liquid of the nanofiltration membranes 2 and 3, and the non-permeated liquid of the reverse osmosis membrane were powdered by spray drying.
• the powder was left on a laboratory table in a room for 1 hour, and then pressed with a finger to evaluate the stickiness of the powder using as an index whether or not the powder adhered to the hand. .
• the permeated liquid of the microfiltration membrane, the permeated liquid of the nanofiltration membranes 2 and 3, and the non-permeated liquid of the reverse osmosis membrane were measured for the concentration of coumaric acid and xylose by the method of Reference Example 4 and Reference Example 5.
• Xylose content (% (w/w)) was measured according to the following formula.
• Xylose content (%) relative to coumaric acid xylose concentration (w/v)/coumaric acid concentration (w/v) x 100
• Table 6 shows the results. As shown in Table 6, by filtering with a nanofiltration membrane having a molecular weight cut off of less than 1000, the permeated liquid of the nanofiltration membrane cannot be visually confirmed to be colored, and the absorption amount by the spectrophotometer increases, resulting in decolorization. I was able to In addition, with the enzyme added, the change in absorption by spectrophotometer after preparation was smaller than that with no enzyme added. As for the dried powder, the one obtained by adding the enzyme was not sticky upon contact.

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